SUMMARY During neural development, trillions of neurites intermingle in a complex environment in which they must recognize and connect with their circuit partners to achieve synaptic specificity. This ability implies that neurons are able to distinguish between each other?a mechanism likely involving cell type-specific molecular cues. One role of these cues is to prevent neurons from connecting with inappropriate partners. The retina is a valuable model system to study neural development due to its accessibility, well-characterized cell types, and laminar organization of inner retinal neuron synapses into a dense neuropil called the inner plexiform layer (IPL). Retinal neurons that share a role in visual processing synapse in discrete IPL sublaminae. If neurons leave their sublayer, or allow the wrong cells to enter their sublayer, they are at risk of wiring with the wrong partners. Thus, the establishment of these lamina-specific connections is critical for visual information processing and for visual perception. We are beginning to understand how retinal circuits come together in specific sublayers; however, it is still unclear how repulsive cues prevent circuits from crosswiring. The objective of this application is to determine how the integrity of one specific retinal circuit, the direction selective (DS) circuit, is established and maintained within the crowded environment of the IPL. The central hypothesis is that mutual repulsion between DS neurons and their neighbors establishes circuit integrity. The rationale for this work is that identifying the molecular mechanisms that mediate repulsion between adjacent retinal circuits will give insight into synaptic specificity in the retina and will likely be relevant for other brain circuits. Three specific aims have been formulated to directly test the central hypothesis: Aim 1) Identify molecule(s) that define the DS circuit IPL layers. Preliminary data suggest starburst dendrites can repel non- DS amacrines. The repulsive molecule FLRT2 is expressed by starburst and DS ganglion cell arbors; this proposal will determine whether it is responsible for this repulsion. Aim 2) Identify molecule(s) expressed by GAD65+ amacrines that exclude them from DS circuit IPL layers. GAD65+ amacrine arbors form sharp boundaries with DS circuit IPL layers. Preliminary data show these cells express Unc5c, a known repulsive receptor. Here it will be determined whether it mediates repulsion from DS circuit arbors. Aim 3) Identify molecule(s) that limit the DS circuit to a specific IPL territory. If DS circuit arbors left their defined sublayers, they could connect with inappropriate partners. The preliminary data suggest that FLRT2 can also function as a receptor to mediate repulsion; this Aim will test this idea. Together, these studies will reveal the specific repulsive interactions between adjacent neural circuits that drive synaptic specificity in the DS circuit. The knowledge gained from this study will serve as a conceptual framework for better understanding the establishment and maintenance of synaptic specificity throughout the CNS. This new information will be applicable to the fields of retinal development as well as neuroregenerative therapies.